Critical events of HIV disease occur in lymphoid tissue. One of such events is HIV-1 divergence into a swarm of viral quasispecies, whose dominant forms change in the course of the disease. In particular, CCR5-utilizing (R5) HIV-1 variants predominate in early stages of HIV-1 disease, whereas CXCR4-utilizing (X4) HIV-1 variants may dominate later. When such a switch occurs, it is followed by an accelerated progression to AIDS. The general aim of this project is to investigate the mechanisms of the R5-X4 switch and of the immunodeficiency development, associated with such a switch. We postulate that R5 and X4 HIV variants affect each other and also that various microbes can selectively inhibit or stimulate R5 or X4 replication by modulating the chemokine/cytokoine network, thus contributing to the switch of viral dominance. We addressed mechanisms of HIV-1 immunopathogenesis in human lymphoid tissue ex vivo that preserves tissue cytoarchitecture and supports productive HIV infection without exogeneous stimulation or activation. These tissues are infected ex vivo with a mixture of HIV-1 quasipsecies differeing in co-receptor usage and with HIV co-pathogens, in particular human herpesvirus 6 (HHV-6). 1. CXCR4-tropic HIV-1 suppresses replication of CCR5-tropic HIV-1 in human lymphoid tissue ex vivo by selective induction of CC-chemokines. To evaluate replication of individual R5- and X4- variants, we developed a new real-time RT PCR-based assay, which accurately estimates the number of viral-specific RNA copies even when their relative abundance is two orders of magnitude. We designed primers that recognize only target sequences of gp120 that specific for R5 and X4 HIV-1 variants, and do not give any non-specific amplification. We verified the accuracy of the assay by comparing the results obtained with viral-specific primers and those obtained with a primer common to all the viruses. We applied this newly developed assay to an ex vivo model consisting of blocks of human lymphoid tissue infected with mixtures of R5 and X4 HIV-1 variants. We used two pairs of HIV-1: one pair consisting of the prototypic X4 and R5 HIV-1 variants, LAV.04 and SF162, respectively, and the other pair consisting of the X4-skewed dualtopic (R5X4) 89.6 and its R5 chimera 89-v345.SF to study whether the R5 and X4 viruses interfere with each other in the context of lymphoid tissue. On average, LAV.04 infection suppressed SF162 replication to a level of 7+/-2% relative to that of tissues infected with SF162 alone. In contrast, the levels of LAV.04 replication in tissues coinfected with SF162 were not different from those in controls infected with LAV.04 only. Also, 89.6 replicated to a level similar to that in singly infected tissue whereas replication of the R5-chimera 89-v345.SF in tissues coinfected with 89.6 was suppressed to a level of 20+/-9% relative to that of matched tissues infected with 89-v345.SF only. To study whether X4 merely prevents the establishment of R5 infection or affects ongoing R5 infection as well, we delayed tissue inoculation with LAV.04 for 3 to 144 h to allow the establishment of SF162 infection. In all experiments, LAV.04 significantly inhibited SF162 replication in coinfected tissues compared with singly infected tissues. Since CC-chemokines are potent inhibitors of replication of R5 HIV-1 variants both in isolated cells and in human lymphoid tissues, we investigated whether X4 infection upregulates CC-chemokines. Using a multiplex Luminex assay, we evaluated the concentrations of a panel of cytokines in the culture medium bathing lymphoid tissue. In SF162-infected tissues, the average concentrations of all measured chemokines/cytokines were similar to those in uninfected controls. In contrast, in tissues infected with LAV.04 either alone or together with SF162, the levels of MIP-1 alpha, MIP-1 beta, and RANTES were increased 4 to 7 fold relative to those in matched uninfected controls. Flow cytometry revealed that X4 infection increases both the number of chemokine producing cells and cell productivity. A cocktail of exogenous CC-chemokines at concentrations similar to those induced by LAV.04 recapitulated the effects of this X4 virus on SF162 replication. In conclusion, we have shown that X4 and R5 HIV-1 variants interfere with each other in human lymphoid tissues and that such interference is associated with an upregulation of CC-chemokines. X4 inhibition of R5 replication may result in a shift of dominance among these HIV-1 variants, thus contributing to the R5-X4 switch. 2. Coinfection with HHV-6 selectively suppresses R5 HIV-1 variants in human lymphoid tissue ex vivo In vivo, disease progression is associated with the emergence of concurrent infections that may affect the course of HIV disease by mechanisms that remain unknown. A lymphotropic agent frequently reactivated in HIV-infected patients is human herpesvirus 6 (HHV-6), which has been proposed as a cofactor in AIDS progression. As multiple interactions have been documented between HHV-6 and HIV-1 both in vitro and in vivo, we investigated whether these two viruses affect each other within the context of human lymphoid tissue ex vivo. Viral strains belonging to both subgroup A (strain GS) and subgroup B (strain PL1) were able to productively infect human tonsillar tissue fragments. The majority of productively infected cells were CD4+ T lymphocytes expressing a memory phenotype (CD45RA-62L-); while infection of CD8+ T cells was efficient only with HHV-6 A. Little, if any, HHV-6 replication was documented in B cells and non-lymphoid cells. Expression of CD4 was upregulated, whereas both CD46, which serves as a cellular receptor for HHV-6, and CD3 were downmodulated. These results provide a novel ex vivo model to investigate the complex interactions between HHV-6 and other pathogens of the immune system, in particular HIV. Strikingly, coinfection with HHV-6 differentially affected the replication of R5 and X4 HIV-1 variants. A dramatic suppression of HIV-1 replication was observed in tissues coinfected with HHV-6 and the CCR5-using variant SF162 (viral replication level was 23+/-5% relative to matched tissues infected by SF162 alone). In contrast, there was a slight enhancement in the replication of the CXCR4-using variant LAV.04 in matched tissue blocks. In agreement with our results with uncloned HIV-1 isolates, HHV-6 coinfection of lymphoid tissue resulted in a marked inhibition of 89-v345.SF replication (viral replication level was 21+/-14% relative to matched tissues not coinfected with HHV-6) and did not affect 89.6 replication (viral replication level was 99+/-43% relative to matched tissues not coinfected with HHV-6). To investigate the potential mechanisms underlying the effects of HHV-6 on HIV-1 infection, we evaluated the production of RANTES, MIP-1 alpha and MIP-1 beta, the three CC chemokines that bind R5. In all the tissues infected with HHV-6, either alone or in combination with HIV-1, we documented a dramatic increase in the production of RANTES only. Adding RANTES to the culture medium in amounts similar to those induced by HHV-6 causes inhibition of R5, but not X4 replication to level similar to that caused by HHV-6 coinfection. Our observations suggest that HHV-6 replication, an event that frequently occurs in vivo during the progression of HIV-1 disease, selectively suppresses CCR5- HIV-1 variants. Thus, it is possible that HHV-6 favors the latter variants in coinfected tissues, from which they can spread throughout the entire organism and become dominant, as it frequently occurs during AIDS progression. Exploitation of the ability of HHV-6 to induce RANTES in lymphoid tissue might lead to novel therapeutic and preventive strategies against HIV infection.